A granzyme B-targeting complex, a radiopharmaceutical, and its imaging applications in tumor immunotherapy.

Abstract

This invention relates to a granzyme B-targeting complex, a radiopharmaceutical, and their imaging applications in tumor immunotherapy, belonging to the field of nuclear medicine diagnostics. The complex is covalently modified, triggering a covalent group proximity effect by recognizing the binding of a peptide to granzyme B, achieving irreversible covalent linkage between the probe and the target protein. This significantly enhances the probe's selective retention at the target site and extends the imaging time window. After radionuclide labeling, the complex can be prepared into a radiopharmaceutical for nuclear medicine imaging; the labeling process is simple and exhibits good stability. This radiopharmaceutical allows for non-invasive, specific, and quantitative monitoring of granzyme B expression levels via PET or SPECT, reflecting the activity status of immune cells in vivo. This invention has significant clinical application value and promising prospects for the early and accurate prediction and dynamic monitoring of tumor immunotherapy efficacy.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine diagnostic and therapeutic technology, and in particular to a granzyme B targeting complex, a radiopharmaceutical, and its imaging application in tumor immunotherapy. Background Technology

[0002] Granzyme B is a protease involved in the process of cytotoxic lymphocyte-induced rapid target cell death. Cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells) release perforin and granzyme upon recognizing target cells. Studies suggest that perforin facilitates granzyme entry into target cells and intracellular localization, thereby inducing cell death. As the most important effector molecule of T cell killing activity, granzyme B is one of the key markers for the activation and cytotoxic effects of CTLs or NK cells.

[0003] Currently, tumor immunotherapy has achieved a series of significant clinical breakthroughs, such as antibodies targeting immune checkpoints (e.g., CTLA-4, PD-1 / PD-L1) and autologous T-cell (CAR-T) therapy based on chimeric antigen receptors. However, the efficacy rate of tumor immunotherapy is currently low. For example, the efficacy rate of anti-PD-1 immune checkpoint inhibition is often less than 30%. Therefore, accurate early prediction of the efficacy of immunotherapy to guide precision treatment is crucial.

[0004] Nuclear medicine molecular imaging technology, with its advantages of being non-invasive, dynamic, and capable of quantitative imaging, provides crucial technical support for disease treatment monitoring. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are representative technologies in this field. Granzyme B expression is closely related to immune responses. Currently, granzyme B-targeting imaging probes face bottlenecks such as scarcity of types, low absolute uptake by tumor tissue, and short in vivo retention time. Covalent modification offers an innovative approach to solving this problem: the recognition peptide first binds to the target protein, and then irreversible covalent linkage is triggered by the proximity effect. Applying this strategy to granzyme B probe modification can improve the selective retention and imaging window of the probe at the target site. Developing novel nuclear medicine imaging drugs with granzyme B target specificity, affinity, and good in vivo metabolism for evaluating and monitoring the efficacy of tumor immunotherapy is of significant practical importance for advancing clinical diagnosis and treatment. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned problems in the prior art and provide a granzyme B-targeting complex, a radiopharmaceutical, and its imaging application in tumor immunotherapy. The complex of this invention, after being labeled with a radionuclide, becomes a radiopharmaceutical for nuclear medicine imaging. Non-invasive and specific monitoring of granzyme B expression levels can be achieved through nuclear medicine PET or SPECT imaging, reflecting the activity of immune cells in vivo. The granzyme B-specific radiopharmaceutical of this invention has a simple preparation and labeling process and features high target site uptake values, and is expected to be widely promoted and clinically applied in the fields of tumor immunotherapy efficacy evaluation, T cell activity evaluation, and autoimmune diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A granzyme B targeting complex has the structure shown in formula (I):

[0007] Wherein, R1 is a covalent reactive group selected from N-hydroxysuccinimide ester, maleimide, iodoacetyl, fluoroacetyl, bromoacetyl, pyridine disulfide, carbodiimide, fluorosulfonyl, acyl azide, sulfonyl chloride, ethylene oxide, and vinyl; the covalent reactive group R1 can specifically covalently bind to amino acid residues in the target molecule, wherein the amino acid residues include at least one of the following: amino groups (–NH) of lysine, histidine, and arginine residues; thiol groups (–SH) of cysteine ​​residues; and hydroxyl groups (–OH) of serine and threonine residues; Linker is a linker selected from straight-chain or branched alkyl, alkylene glycol, or polyethylene glycol (PEG) segments. n (n=1~20). R2 is a bifunctional chelating group selected from DOTA, NOTA, HYNIC, MAG2, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, BAPEN, Df, DFO, TACN, NO2A, NOTAM, CB-DO2A, Cyclen, DO3A, DO3AP, MAS3, MAG3, or groups formed from isonitriles.

[0008] The R2 is selected from the group shown in formula (ii) or formula (iii):

[0009] The Linker is selected from any one of the groups shown in formulas (iv) to (xi), where m and n are independently integers from 0 to 9:

[0010] The R1 is selected from any one of the groups shown in Formula (XII) to Formula (XIX):

[0011] A radiopharmaceutical comprising the granzyme B targeting complex and a radionuclide labeled with the granzyme B targeting complex.

[0012] According to the present invention, the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide.

[0013] The radionuclides are selected from 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 99m Tc, 11 C 123 I, 125 I, 124 I, 177 Lu、 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0014] Specifically, the diagnostic radionuclide is preferably... 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I. The therapeutic radionuclide is preferably... 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0015] Preferably, the radionuclide is selected from... 68 Ga、 64 Cu、 177 Lu、 18 Any one of F.

[0016] This invention provides a method for preparing a granzyme B-targeted radiopharmaceutical, comprising the following steps: dissolving a granzyme B-targeting complex in a radiolabeled buffer solution, then adding different radionuclides to react, and after the reaction, separating and purifying the reaction solution using a Sep-Pak C18 chromatographic column to obtain the corresponding granzyme B-targeted radiopharmaceutical.

[0017] According to a specific embodiment of the present invention, when the complex is a DOTA-coupled complex, the radionuclide is... 68 Ga、 64 Cu、 111 In、 86 Y or 177 When any one of Lu is present, the preparation method includes the following steps: dissolving the DOTA-conjugated complex in an acidic buffer solution, and then adding... 68 GaCl3, 64 CuCl2, 111 InCl3 or 86 YCl3 was reacted at 35-95℃ for 10-60 min. The reaction solution was then purified by Sep-Pak C18 column chromatography to obtain the corresponding... 68 Ga、 64 Cu、 111 In or 86 Y-labeled complexes.

[0018] According to a specific embodiment of the present invention, when the complex is a NOA-coupled complex, the radionuclide is... 68 Ga、 64 Cu、 18 When any of F is present, the preparation method includes the following steps: dissolving the NOTA-coupled complex in an acidic buffer solution, and then adding... 68 GaCl3, 64 The reaction solution was incubated with CuCl2 at 35–95 °C for 10–30 min. After cooling, the reaction solution was purified by Sep-Pak C18 chromatography to obtain the corresponding... 68 Ga or 64Cu-labeled complexes; or, 18 F ions were mixed with AlCl3 in sodium acetate buffer and reacted at room temperature for 2–8 min. Then, the Nota-coupled complex was added to the mixture and reacted at 105–115 °C for 10–20 min. After cooling, the reaction solution was purified by Sep-Pak C18 column chromatography to obtain the corresponding... 18 F-labeled complexes.

[0019] According to a specific embodiment of the present invention, when the complex is a MAG2 coupled complex or a HYNIC coupled complex, the radionuclide is... 99m At Tc, the preparation method includes the following steps: dissolving the MAG2 coupled complex in ammonium acetate and tartrate buffer, and then adding Na 99m TcO4 was mixed thoroughly, and then freshly prepared SnCl2 was added. The mixture was then heated to 95–105 °C and reacted for 40–80 min. After cooling, the reaction solution was purified by Sep-Pak C18 chromatography to obtain the corresponding... 99m Tc-labeled complexes; or, the HYNIC-coupled complex, TPPTS succinate buffer, and tricine succinate buffer are mixed and then Na is added. 99m TcO4 was then heated to 95-105℃ for 20-40 min. After cooling, the reaction solution was purified by Sep-Pak C18 chromatography to obtain the corresponding... 99m Tc-labeled complexes.

[0020] According to the present invention, preferably after the labeled complexes are separated and purified, the purified product is diluted with physiological saline and then sterile filtered to obtain injection solutions of each complex.

[0021] The application of the granzyme B targeting complex or the radiopharmaceutical in the preparation of nuclear medicine imaging reagents.

[0022] The nuclear medicine imaging reagents are used for monitoring the efficacy of tumor immunotherapy and / or evaluating T-cell activity. The tumor immunotherapy includes anti-PD-1 / PD-L1 immune checkpoint inhibitor therapy, anti-CTLA-4 immune checkpoint inhibitor therapy, CAR-T cell immunotherapy, and CAR-NK cell immunotherapy.

[0023] Specifically, targeted imaging of the dynamic expression level of granzyme B in the tumor microenvironment can accurately reflect the activation status of cytotoxic lymphocytes (CTL, NK cells): in the treatment of immune checkpoint inhibitors such as anti-PD-1 / PD-L1 and anti-CTLA-4, the enrichment of granzyme B can be quantitatively detected to predict the treatment response; in cellular immunotherapy such as CAR-T and CAR-NK, the infiltration and functional activity of effector cells in the tumor site can be tracked in real time to distinguish between "pseudo-progression" and true treatment response.

[0024] The novel granzyme B probe in this invention, with its high target retention due to covalent modification, can achieve more accurate and longer-lasting imaging in the above-mentioned scenarios, thereby improving the accuracy of disease diagnosis and treatment assessment and extending the time window.

[0025] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0026] 1. The granzyme B probe (i.e. granzyme B targeting complex) of the present invention has a granzyme B recognition backbone and a covalent module, and has good chemical stability, biosafety and biodistribution properties. The preparation method is simple and easy, and it can be used for PET / SPECT imaging of immune-related diseases.

[0027] 2. The granzyme B probe of the present invention has good imaging effect on the dynamic expression level of granzyme B in the tumor microenvironment, which can reflect the activity of immune cells, dynamically monitor the activity of immune cells, and evaluate the effect of immunotherapy. Attached Figure Description

[0028] Figure 1 This is the mass spectrum of the NOA-conjugated covalent granzyme B probe GB-F3.

[0029] Figure 2 This is the mass spectrum of the NOA-conjugated covalent granzyme B probe GB-F4.

[0030] Figure 3 for 68 In vitro stability results of Ga-labeled covalent granzyme B probe GB-F4 in PBS.

[0031] Figure 4 for 68 In vitro stability results of Ga-labeled covalent granzyme B probe GB-F4 in FBS.

[0032] Figure 5 for 68 Pharmacokinetic results of Ga-labeled covalent granzyme B probe GB-F4.

[0033] Figure 6 for 68Experimental results of PET imaging of Ga-labeled covalent granzyme B probes GB-F3, GB-F4, and GB-F5 in MC38 tumor-bearing mice at 0.5h, 1h, and 2h.

[0034] Figure 7 for 68 Quantitative circle values ​​of Ga-labeled covalent granzyme B probes GB-F3, GB-F4, and GB-F5 in PET imaging at 30 min, 60 min, and 120 min in MC38 tumor-bearing mice.

[0035] Figure 8 for 68 Experimental results of 60 min PET imaging of Ga-labeled covalent granzyme B probe GB-F4 in MC38 tumor-bearing mice.

[0036] Figure 9 for 68 A graph showing the correlation between the quantitative circle value of the Ga-labeled covalent granzyme B probe GB-F4 in MC38 tumor-bearing mice after 60 min PET imaging and the expression level of granzyme B in the tumor as measured by the corresponding in vitro ELISA.

[0037] Figure 10 for 68 Results of PET imaging experiments monitoring different stages of anti-PD-1 immunotherapy in mice using Ga-labeled covalent granzyme B probe GB-F4. Detailed Implementation

[0038] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Example 1

[0039] Synthesis of granzyme B targeting complexes.

[0040] The granzyme B-targeting complex GB-F3 was synthesized according to the following synthetic route:

[0041] The reaction conditions included: (a) CTC resin, DIPEA, and DCM; (b) a 20% piperidine DMF solution; (c) a DMF solution of glutamic acid-N-Fmoc-γ-tert-butyl ester, HBTU, HOBt, and DIPEA, and a 20% piperidine DMF solution; (d) a DMF solution of Fmoc-N-isoleucine, HBTU, HOBt, and DIPEA, and a 20% piperidine DMF solution; and (e) Fmoc-N-diethylene glycol-carboxylic acid, HBTU, and HOBt. (f) DMF solution of DIPEA and 20% piperidine; (g) DMF solution of Nota-succinimide ester and DIPEA; (h) DMF solution of hexafluoroisopropanol; (i) DMF solution of Nα-fluorenemethoxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine, HBTU, HOBt and DIPEA; (ii) DMF solution of 2-[(methylamino)methyl]but-3-enamide, HBTU, HOBt and DIPEA, HCl-Dioxane.

[0042] Synthesis of Compound 2: 1.00 g of CTC resin was placed in a solid-phase synthesis tube. 2 mL of dichloromethane (DCM) was added, and the mixture was shaken at room temperature for 5 min to swell. The solution was then filtered and discarded. This swelling process was repeated three times. The resin was then washed three times with N,N-dimethylformamide (DMF) and dried. Compound 1 (111.21 mg, 0.33 mmol, molar ratio to resin 1:3.3) was dissolved in 2 mL of a mixed solvent (V(DCM):V(DMF) = 1:1). N,N-diisopropylethylamine (DIPEA, 78 mg, 0.6 mmol) was added and stirred to dissolve the compound. The mixture was transferred to a solid-phase synthesis tube and stirred at room temperature for 2 h (TLC monitoring showed complete consumption of Compound 1). After the reaction was completed, the resin was washed three times with 2 mL of DCM (5 min each time) and dried under vacuum. Then, 7 mL of blocking mixture (V(DCM):V(MeOH):V(DIPEA)=10:10:1) was added, and the unreacted active sites of the resin were blocked by shaking (5 min each time, repeated 3 times). The resin was then washed three times with 2 mL of DCM (5 min each time), and the solvent was dried under reduced pressure to obtain CTC resin loaded with compound 2.

[0043] Synthesis of Compound 3: The coupling of amino acids was performed according to the standard Fmoc solid-phase synthesis method. A certain mass of Compound 2 (0.25 mmol) was placed in a 10 mL solid-phase synthesis tube, and 2 mL of dichloromethane (DCM) was added to swell the mixture. This process was repeated three times, 5 min each time. The mixture was then washed three times with N,N-dimethylformamide (DMF), 5 min each time. The amino protecting group of Fmoc was removed using a DMF solution (v / v) containing 20% ​​piperidine.

[0044] Glutamic acid-N-FMOC-γ-tert-butyl ester (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0045] Fmoc-N-isoleucine (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and after washing with DMF, the mixture was dried under vacuum to obtain compound 3.

[0046] Synthesis of Compound 4: Fmoc-N-diethylene glycol-carboxylic acid (0.06 mmol) and HOBt (0.06 mmol) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol) was added for activation for 5 min. Then, DIPEA (0.144 mmol) was added and mixed thoroughly. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) after the reaction and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0047] Synthesis of Compound 5: 0.06 mmol of NOA-succinimide ester was mixed with 0.144 mmol of DIPEA; the mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. The resin was then washed three times (2 min each time) with 2 mL of DMF and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, followed by washing with DMF and drying under vacuum.

[0048] Synthesis of Compound 6: 2 mL of dichloromethane (DCM) was added to the synthesis tube containing Compound 5 and the resin was washed three times (5 min each time), then dried under vacuum. 3 mL of a cutting mixture (V(HFIP):V(DCM) = 2:8, i.e., 20% HFIP / DCM) was added to the synthesis tube, ensuring the liquid level was approximately 1 cm above the resin. The reaction was carried out with shaking at room temperature for 1 h. After the reaction was complete, the supernatant was collected by filtration. The supernatant was transferred to a rotary evaporator flask and concentrated under reduced pressure to remove DCM and HFIP. After concentration, 5 mL of cold diethyl ether was added to precipitate the product. The precipitate was collected by centrifugation (3000 rpm, 5 min) and dried under vacuum to obtain Compound 6.

[0049] Synthesis of compound 7: Compound 6 (0.1 mmol) was dissolved in 5 mL of anhydrous DMF, and Nα-fluorenylmethoxycarbonyl-Nε-tert-butyloxycarbonyl-L-lysine (0.12 mmol), HBTU (0.15 mmol), HOBt (0.15 mmol), and DIPEA (0.3 mmol) were added sequentially. The mixture was stirred at room temperature for 1 h, and after precipitation with cold diethyl ether, it was centrifuged to obtain compound 7.

[0050] Synthesis of GB-F3: In a clean flask, compound 7 (0.1 mmol), 2-[(methylamino)methyl]but-3-enamide (0.12 mmol), HBTU (0.15 mmol), HOBt (0.15 mmol), and DIPEA (0.3 mmol) were stirred at room temperature for 1 h. The mixture was extracted with ethyl acetate, and the organic phase was dried, concentrated, and purified. 1 M HCl-Dioxane was added, and the mixture was reacted for 30 min, followed by rotary evaporation to concentrate. After precipitation with cold diethyl ether, the mixture was centrifuged to obtain GB-F3. (See [link to relevant documentation]). Figure 1 . Example 2

[0051] The granzyme B targeting complex GB-F4 was synthesized according to the following synthetic route.

[0052]

[0053] The reaction conditions included: (a) CTC resin, DIPEA, and DCM; (b) a 20% piperidine DMF solution; a DMF solution of Fmoc-N-proline, HBTU, HOBt, and DIPEA; (c) a 20% piperidine DMF solution; a DMF solution of glutamic acid-N-FMOC-γ-tert-butyl ester, HBTU, HOBt, and DIPEA; and (d) a 20% piperidine DMF solution; a DMF solution of Fmoc-N-isoleucine, HBTU, HOBt, and DIPEA. (e) DMF solutions of Fmoc-N-diethylene glycol-carboxylic acid, HBTU, HOBt and DIPEA, and DMF solutions of 20% piperidine; (f) DMF solutions of Nota-succinimide ester and DIPEA; (g) DCM solutions of hexafluoroisopropanol; (h) Isobutyl chloroformate, diazomethane, N-methylmorpholine, anhydrous tetrahydrofuran, and HCl-Dioxane; (i) HCl-Dioxane.

[0054] Synthesis of Compound 2: 1.00 g of CTC resin was placed in a solid-phase synthesis tube. 2 mL of dichloromethane (DCM) was added, and the mixture was shaken at room temperature for 5 min to swell. The solution was then filtered and discarded. This swelling process was repeated three times. The resin was then washed three times with N,N-dimethylformamide (DMF) and dried. Compound 1 (135.64 mg, 0.33 mmol, molar ratio to resin 1:3.3) was dissolved in 2 mL of a mixed solvent (V(DCM):V(DMF) = 1:1). N,N-diisopropylethylamine (DIPEA, 78 mg, 0.6 mmol) was added and stirred to dissolve the compound. The mixture was transferred to a solid-phase synthesis tube and stirred at room temperature for 2 h (TLC monitoring showed complete consumption of Compound 1). After the reaction was completed, the resin was washed three times with 2 mL of DCM (5 min each time) and dried under vacuum. Then, 7 mL of blocking mixture (V(DCM):V(MeOH):V(DIPEA)=10:10:1) was added, and the unreacted active sites of the resin were blocked by shaking (5 min each time, repeated 3 times). The resin was then washed three times with 2 mL of DCM (5 min each time), and the solvent was dried under reduced pressure to obtain CTC resin loaded with compound 2.

[0055] Synthesis of Compound 3: The coupling of amino acids was performed according to the standard Fmoc solid-phase synthesis method. A certain mass of Compound 2 (0.25 mmol) was placed in a 10 mL solid-phase synthesis tube, and 2 mL of dichloromethane (DCM) was added to swell the mixture. This process was repeated three times, 5 min each time. The mixture was then washed three times with N,N-dimethylformamide (DMF), 5 min each time. The amino protecting group of Fmoc was removed using a DMF solution (v / v) containing 20% ​​piperidine.

[0056] Fmoc-N-proline (0.06 mmol, 3 eq. relative to the resin active site) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF. HBTU (0.072 mmol, 3.6 eq.) was added and the mixture was stirred for 5 min to activate the resin. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed thoroughly. The mixture was transferred to a synthesis tube and stirred electromagnetically at room temperature for 1 h (the reaction was monitored for completeness using the ninhydrin method). After the reaction, the resin was washed three times with 2 mL of DMF (2 min each time) and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, followed by washing with DMF and drying under vacuum.

[0057] Glutamic acid-N-FMOC-γ-tert-butyl ester (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0058] Fmoc-N-isoleucine (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and after washing with DMF, the mixture was dried under vacuum to obtain compound 3.

[0059] Synthesis of Compound 4: Fmoc-N-diethylene glycol-carboxylic acid (0.06 mmol) and HOBt (0.06 mmol) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol) was added for activation for 5 min. Then, DIPEA (0.144 mmol) was added and mixed thoroughly. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) after the reaction and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0060] Synthesis of Compound 5: 0.06 mmol of NOA-succinimide ester was mixed with 0.144 mmol of DIPEA; the mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. The resin was then washed three times (2 min each time) with 2 mL of DMF and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, followed by washing with DMF and drying under vacuum.

[0061] Synthesis of Compound 6: 2 mL of dichloromethane (DCM) was added to the synthesis tube containing Compound 5 and the resin was washed three times (5 min each time), then dried under vacuum. 3 mL of a cutting mixture (V(HFIP):V(DCM) = 2:8, i.e., 20% HFIP / DCM) was added to the synthesis tube, ensuring the liquid level was approximately 1 cm above the resin. The reaction was carried out with shaking at room temperature for 1 h. After the reaction was complete, the supernatant was collected by filtration. The supernatant was transferred to a rotary evaporator flask and concentrated under reduced pressure to remove DCM and HFIP. After concentration, 5 mL of cold diethyl ether was added to precipitate the product. The precipitate was collected by centrifugation (3000 rpm, 5 min) and dried under vacuum to obtain crude Compound 6.

[0062] Synthesis of Compound 7: Compound 6 (0.1 mmol) was dissolved in 5 mL of anhydrous tetrahydrofuran (THF) and placed in an ice bath at 0 °C. N-methylmorpholine (NMM, 0.12 mmol, 1.2 eq.) was added sequentially and stirred for 5 min, followed by the addition of isobutyl chloroformate (IBCF, 0.12 mmol, 1.2 eq.), and stirring was continued for 10 min. A diethyl ether solution of diazomethane (0.5 M, 0.2 mmol, 2.0 eq.) was slowly added dropwise to the above system, and the reaction was maintained at 0 °C for 30 min. The temperature was then raised to room temperature (25 °C) and stirred for 3 h (TLC monitoring showed complete substrate consumption). Excess diazomethane was quenched by adding saturated ammonium chloride solution (2 mL), and the mixture was extracted with ethyl acetate (3 × 5 mL). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and then 2 mL of 1 M HCl / 1,4-dioxane solution was added. The mixture was stirred at room temperature for 30 min, the solvent was removed by rotary evaporation, and the precipitate was precipitated by cold diethyl ether and centrifuged to obtain Compound 7.

[0063] Synthesis of GB-F4: In a clean flask, add compound 7, then add 2 mL of 2M HCl / 1,4-dioxane solution. Stir at room temperature for 1 h, remove the solvent by rotary evaporation, precipitate with cold diethyl ether, and centrifuge to obtain GB-F4. (See [link to relevant documentation]). Figure 2 .

[0064] Example 3

[0065] Reaction conditions: (a) CTC resin, DIPEA, DCM; (b) 20% piperidine in DMF solution; (c) Glutamic acid-N-FMOC-γ-tert-butyl ester, HBTU, HOBt and DIPEA in DMF solution; (d) 20% piperidine in DMF solution, Fmoc-N-isoleucine, HBTU, HOBt and DIPEA in DMF solution, 20% piperidine in DMF solution; (e) Fmoc-N-diethylene glycol (f) DMF solutions of alcohol-carboxylic acids, HBTU, HOBt and DIPEA, and 20% piperidine; (g) DMF solutions of Nota-succinimide ester and DIPEA; (h) DMF solutions of hexafluoroisopropanol; (i) DMF solutions of Nα-fluorenemethoxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine, HBTU, HOBt and DIPEA; and (v) HCl-Dioxane, m-CPBA and DCM.

[0066] Synthesis of Compound 2: 1.00 g of CTC resin was placed in a solid-phase synthesis tube. 2 mL of dichloromethane (DCM) was added, and the mixture was shaken at room temperature for 5 min to swell. The solution was then filtered and discarded. This swelling process was repeated three times. The resin was then washed three times with N,N-dimethylformamide (DMF) and dried. Compound 1 (111.21 mg, 0.33 mmol, molar ratio to resin 1:3.3) was dissolved in 2 mL of a mixed solvent (V(DCM):V(DMF) = 1:1). N,N-diisopropylethylamine (DIPEA, 78 mg, 0.6 mmol) was added and stirred to dissolve the compound. The mixture was transferred to a solid-phase synthesis tube and stirred at room temperature for 2 h (TLC monitoring showed complete consumption of Compound 1). After the reaction was completed, the resin was washed three times with 2 mL of DCM (5 min each time) and dried under vacuum. Then, 7 mL of blocking mixture (V(DCM):V(MeOH):V(DIPEA)=10:10:1) was added, and the unreacted active sites of the resin were blocked by shaking (5 min each time, repeated 3 times). The resin was then washed three times with 2 mL of DCM (5 min each time), and the solvent was dried under reduced pressure to obtain CTC resin loaded with compound 2.

[0067] Synthesis of Compound 3: The coupling of amino acids was performed according to the standard Fmoc solid-phase synthesis method. A certain mass of Compound 2 (0.25 mmol) was placed in a 10 mL solid-phase synthesis tube, and 2 mL of dichloromethane (DCM) was added to swell the mixture. This process was repeated three times, 5 min each time. The mixture was then washed three times with N,N-dimethylformamide (DMF), 5 min each time. The amino protecting group of Fmoc was removed using a DMF solution (v / v) containing 20% ​​piperidine.

[0068] Glutamic acid-N-FMOC-γ-tert-butyl ester (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0069] Fmoc-N-isoleucine (0.06 mmol, 3 eq.) and HOBt (0.06 mmol, 3 eq.) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol, 3.6 eq.) was added for activation for 5 min. Then, DIPEA (0.144 mmol, 7.2 eq.) was added and mixed well. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and after washing with DMF, the mixture was dried under vacuum to obtain compound 3.

[0070] Synthesis of Compound 4: Fmoc-N-diethylene glycol-carboxylic acid (0.06 mmol) and HOBt (0.06 mmol) were dissolved in 2 mL of DMF, and HBTU (0.072 mmol) was added for activation for 5 min. Then, DIPEA (0.144 mmol) was added and mixed thoroughly. The mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the solution was dried under vacuum. The resin was washed three times with 2 mL of DMF (2 min each time) after the reaction and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, and the solution was washed with DMF and dried under vacuum.

[0071] Synthesis of Compound 5: 0.06 mmol of NOA-succinimide ester was mixed with 0.144 mmol of DIPEA; the mixture was transferred to a synthesis tube and stirred at room temperature for 1 h. After washing with DMF, the mixture was dried under vacuum. The resin was then washed three times (2 min each time) with 2 mL of DMF and dried under vacuum. Fmoc was removed with 20% piperidine / DMF solution, followed by washing with DMF and drying under vacuum.

[0072] Synthesis of Compound 6: 2 mL of dichloromethane (DCM) was added to the synthesis tube containing Compound 5 and the resin was washed three times (5 min each time), then dried under vacuum. 3 mL of a cutting mixture (V(HFIP):V(DCM) = 2:8, i.e., 20% HFIP / DCM) was added to the synthesis tube, ensuring the liquid level was approximately 1 cm above the resin. The reaction was carried out with shaking at room temperature for 1 h. After the reaction was complete, the supernatant was collected by filtration. The supernatant was transferred to a rotary evaporator flask and concentrated under reduced pressure to remove DCM and HFIP. After concentration, 5 mL of cold diethyl ether was added to precipitate the product. The precipitate was collected by centrifugation (3000 rpm, 5 min) and dried under vacuum to obtain crude Compound 6.

[0073] Synthesis of compound 7: Compound 6 (0.1 mmol) was dissolved in 5 mL of anhydrous DMF, and Nα-fluorenylmethoxycarbonyl-Nε-tert-butyloxycarbonyl-L-lysine (0.12 mmol), HBTU (0.15 mmol), HOBt (0.15 mmol), and DIPEA (0.3 mmol) were added sequentially. The mixture was stirred at room temperature for 1 h, and after precipitation with cold diethyl ether, it was centrifuged to obtain compound 7.

[0074] Synthesis of GB-F5: In a clean flask, compound 7 (0.1 mmol) was dissolved in anhydrous dichloromethane (DCM) and cooled in an ice bath at 0 °C. m-CPBA (0.12 mmol, 1.2 eq.) was slowly added, and the mixture was stirred for 1 h. The reaction was then quenched with saturated NaHCO3 solution, extracted with ethyl acetate, and purified by drying and concentrating the organic phase. 1 M HCl-Dioxane was added, and the mixture was reacted for 30 min, followed by rotary evaporation to concentrate the solution. After precipitation with cold diethyl ether, the precipitate was centrifuged to obtain GB-F5. Example 4

[0075] Synthesis of NOTA / DOTA coupled complexes and their application 18 F, 68 Ga、 64 Cu、 111 In、 86 Y、 177 The labeling of any radionuclide with Lu is used to prepare the corresponding radiopharmaceutical. Specifically, the steps include: 68 Ga、 64 Cu、 111 In、 86 Y-labeled radioactive material: Dissolve 50 μg of GB-F4 in 500 μL of 0.1 mol / L sodium acetate buffer (pH 5.5) to prepare the substrate solution. Add 185 MBq of radioactive isotope chloride stock solution (optional) to the above solution. 68 GaCl3, 64 CuCl2, 111 InCl3 or 86(Any of YCl3) was placed at 37℃ for 30 min. After the reaction, the reaction solution was separated and purified using a Sep-Pak C18 column; the purified target product was collected, diluted with physiological saline, and then sterile filtered to obtain the corresponding radionuclide-labeled NOTA conjugate injection solution. 68 Ga marker, 64 Cu labeling, 111 In mark or 86 (Y mark).

[0076] 18 F radioactive labeling: 185–740 MBq 18 F - The solution was co-dissolved with 12 nmol AlCl3 in 100 μL of 0.1 mol / L sodium acetate buffer (pH=4.0) and reacted at room temperature for 5 min. After the reaction was complete, 50 μg of GB-F4 was added to the system, and the temperature was raised to 110 °C and held for 15 min. After the reaction solution cooled to room temperature, it was purified by separation using a Sep-Pak C18 column. The purified fraction was collected, diluted with physiological saline, and then sterilely filtered to obtain the final product. 18 F-labeled complex injection solution.

[0077] 177 Lu radiolabeling: Weigh 50 μg of DOTA-modified GB-F4, add 500 μL of 0.1 mol / L sodium acetate buffer (pH 5.2), vortex for 30 s until completely dissolved, to obtain the stock solution of compound 8. Add 185–740 MBq of [unspecified substance] to the above stock solution. 177 After gently mixing the LuCl3 solution, react it in a 95℃ constant temperature metal bath for 15-30 min. After the reaction is complete, cool the reaction solution to room temperature and slowly load it onto a pretreated Sep-Pak C18 column; first rinse the column with 5 mL of ultrapure water to remove uncomplexed free radicals. 177 Lu 3+ After removing buffer impurities, the target product was eluted with 3 mL of 80% ethanol solution, and the eluent was collected. The collected eluent was transferred to a sterile centrifuge tube, diluted with an appropriate amount of sterile physiological saline (final ethanol concentration ≤10%), and then filtered through a 0.22 μm sterile filter membrane into a sterile injection bottle to obtain the final product. 177 Lu-labeled DOTA-GB-F4 injection. Example 5

[0078] 68 In vitro stability of Ga-labeled Nota complex GB-F4.

[0079] Take 3.7 MBq 68The Ga-labeled Nota complex GB-F4 was dissolved in PBS and FBS and incubated at room temperature for 0 h, 0.5 h, 1 h, and 2 h, respectively, before being connected to a radiation detector by HPLC. The results are shown in... Figure 3 , Figure 4 .visible 68 Ga-labeled NOTA complexes exhibited good stability in both PBS and FBS. Example 6

[0080] 68 Pharmacokinetics of Ga-labeled Nota complex GB-F4.

[0081] Take 7.4 MBq 68 Ga-labeled Nota complex GB-F4 was dissolved in PBS and injected intravenously into C57 tumor-bearing mice. Blood samples were collected from the orbital cavity at 1 min, 5 min, 10 min, 15 min, 30 min, 60 min, and 120 min, and placed in EP tubes. The radioactivity count of each tube was recorded using a γ-count instrument. Pharmacokinetic curves were fitted using DAS software, and the following parameters were calculated: compartment model parameters: half-life (t1 / 2α, t1 / 2β), clearance (CL), apparent volume of distribution (Vd), and area under the curve (AUC0-t, AUC0-∞). The results are shown in [Figure / Table / Insert Table ...Insert Table / Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / Insert Table / In Figure 5 . Example 7

[0082] 68 PET images of Ga-labeled Nota complexes GB-F3, GB-F4, and GB-F5 at multiple time points in MC38 tumor-bearing mice. To compare the advantages of the granzyme B targeting complex described in this invention for in vivo nuclear medical imaging, three structurally similar granzyme B targeting complexes were prepared using a similar synthetic method. All were coupled with Nota before imaging. 68 Ga labeling yielded the corresponding granzyme B-targeted radioactive compound. 7.4 MBq of freshly prepared... 68 Ga-labeled complexes were injected via tail vein into female C57 mice bearing MC38 tumors. Small animal PET / CT imaging was performed 0.5 h, 1 h, and 2 h after injection, followed by isoflurane anesthesia. The imaging results are shown in... Figure 6 The quantitative cyclone results are shown in Figure 7 As can be seen, the present invention 68 Ga-labeled Nota complex GB-F4 exhibits optimal tumor granzyme B uptake and retention time compared to two other contrasting radiopharmaceuticals. Example 8

[0083] 681-hour PET imaging characteristics of Ga-labeled Nota complex GB-F4 in tumor-bearing mice.

[0084] To compare the in vivo nuclear medical imaging advantages of the granzyme B targeting complex GB-F4 described in this invention, it was performed after conjugation with NOA. 68 Ga labeling yielded the corresponding granzyme B-targeted radioactive compound GB-F4. 7.4 MBq of freshly prepared... 68 Ga-labeled complexes were injected via tail vein into female C57 mice bearing MC38 tumors. One hour after injection, the mice were anesthetized with isoflurane, and small animal PET / CT imaging was performed. The imaging results are shown in... Figure 8 As can be seen, the present invention 68 Ga-labeled NOTA complex GB-F4 has a wide imaging window and can reflect the immune level of tumor sites in different individual mouse models. Example 9

[0085] 68 Quantitative analysis of tumor granzyme B expression using PET imaging of Ga-labeled Nota complex GB-F4.

[0086] Take 7.4 MBq of freshly prepared 68 A Ga-labeled complex was injected via tail vein into female C57 mice bearing MC38 tumors in the right axilla. One hour after injection, the mice were anesthetized with isoflurane and subjected to small animal PET / CT imaging. After imaging, the mice were sacrificed, and tumor tissue was harvested. Proteins were extracted from the tumor tissue, and the expression level of granzyme B within the tumor was measured by ELISA. The results are shown in... Figure 9 ,visible 68 The uptake of Ga-labeled DOTA complexes in MC38 tumors showed a good linear correlation with the expression level of granzyme B in tumors as measured by in vitro ELISA. This suggests that the expression level of granzyme B can be non-invasively measured using nuclear medicine imaging of granzyme B-targeted radiopharmaceuticals as described in this invention. Example 10

[0087] 68 Ga-labeled Nota complex GB-F4 can reflect changes in the immune status of mice.

[0088] Female C57 mice with MC38 tumors in their right axilla were injected with 200 μg of anti-PD-1 antibody on days 0, 3, and 6 of the experiment, and the mice were injected with anti-PD-1 antibody on days 0, 3, 6, and 9 of the experiment. 68 PET imaging of Ga-labeled Nota complexes, the results of which are shown in Figure 10 PET imaging of tumor-bearing mice showed significantly increased uptake at the tumor site, indicating that the immune system of tumor-bearing mice was activated and granzyme B secretion was increased.68 Ga-labeled Nota complexes can accurately and dynamically reflect this immune change.

[0089] This invention can be applied to nuclear medicine imaging diagnosis, treatment process monitoring, and efficacy evaluation of tumors and immune abnormalities, providing core technical support for the clinical translation of nuclear medicine precision diagnosis and treatment technologies.

[0090] The various embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments.

Claims

1. A granzyme B targeting complex, characterized in that, It has the structure shown in equation (i): Wherein, R1 is a covalent reactive group selected from N-hydroxysuccinimide ester, maleimide, iodoacetyl, fluoroacetyl, bromoacetyl, pyridine disulfide, carbodiimide, fluorosulfonyl, acyl azide, sulfonyl chloride, ethylene oxide, and vinyl. Linker is a linker selected from straight-chain or branched alkyl, alkylene glycol, or polyethylene glycol segments; R2 is a bifunctional chelating group selected from DOTA, NOTA, HYNIC, MAG2, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, BAPEN, Df, DFO, TACN, NO2A, NOTAM, CB-DO2A, Cyclen, DO3A, DO3AP, MAS3, MAG3, or groups formed from isonitriles.

2. The granzyme B targeting complex as described in claim 1, characterized in that, The R2 is selected from the group shown in formula (ii) or formula (iii): 。 3. The granzyme B targeting complex as described in claim 1, characterized in that, The Linker is selected from any one of the groups shown in formulas (iv) to (xi), where m and n are independently integers from 0 to 9: 。 4. The granzyme B targeting complex as described in claim 1, characterized in that, The R1 is selected from any one of the groups shown in Formula (XII) to Formula (XIX): 。 5. A radiopharmaceutical, characterized in that: The invention comprises the granzyme B targeting complex according to any one of claims 1 to 4, and a radionuclide, wherein the radionuclide labels the granzyme B targeting complex.

6. The radiopharmaceutical as described in claim 5, characterized in that, The radionuclides are selected from 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 99m Tc, 11 C 123 I, 125 I, 124 I, 177 Lu、 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

7. The radiopharmaceutical as described in claim 6, characterized in that: The radionuclides are selected from 68 Ga、 64 Cu、 177 Lu、 18 Any one of F.

8. The use of the granzyme B targeting complex according to any one of claims 1 to 4 or the radiopharmaceutical according to any one of claims 5 to 7 in the preparation of nuclear medicine imaging reagents.

9. The application as described in claim 8, characterized in that: Application of the nuclear medicine imaging reagent in monitoring the efficacy of tumor immunotherapy and / or evaluating T cell activity.

10. The application as described in claim 9, characterized in that: The tumor immunotherapy includes anti-PD-1 / PD-L1 immune checkpoint inhibitor therapy, anti-CTLA-4 immune checkpoint inhibitor therapy, CAR-T cell immunotherapy, and CAR-NK cell immunotherapy.

Images